Prenylated Bibenzyls from the Chinese Liverwort Radula constricta

Article Cite This: J. Nat. Prod. XXXX, XXX, XXX−XXX

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Prenylated Bibenzyls from the Chinese Liverwort Radula constricta and Their Mitochondria-Derived Paraptotic Cytotoxic Activities Chun-Yang Zhang,†,§ Yun Gao,†,§ Rong-Xiu Zhu,‡ Ya-Nan Qiao,† Jin-Chuan Zhou,⊥ Jiao-Zhen Zhang,† Yi Li,† Si-Wen Li,† Sheng-Hua Fan,† and Hong-Xiang Lou*,† †

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Department of Natural Products Chemistry, Key Lab of Chemical Biology of the Ministry of Education, Shandong University, Jinan 250012, People’s Republic of China ‡ School of Chemistry and Chemical Engineering, Shandong University, Jinan 250010, People’s Republic of China ⊥ School of Pharmacy, Linyi University, Linyi 276000, People’s Republic of China S Supporting Information *

ABSTRACT: Nine new prenylated bibenzyls, radstrictins A−I (1− 9), and 11 known congeners were obtained from the Chinese liverwort Radula constricta. Their structures were identified by analysis of HRMS, NMR, and electronic circular dichroism data. Radstrictins A−F (1−6) were isolated as a racemate or scalemic mixtures. All the isolated compounds were subjected to cytotoxicity assessment. Methyl 2,4-dihydroxy-3-(3-methyl-2-butenyl)-6-phenethylbenzoate (10) exhibited significant activity against human lung cancer cell lines A549 and NCI-H1299 with IC50 values of 6.0 and 5.1 μM, respectively. Further research revealed that cell death triggered by 10 occurred via mitochondria-derived paraptosis.

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lines, along with accumulation of numerous cytoplasmic vacuoles. Further investigation revealed that 10 induced cancer cell death through mitochondria-derived paraptosis. Herein, the experimental details pertaining to the isolation, structural determination, and cytotoxicity assessment of the obtained compounds, as well as the mitochondria-derived paraptosis induced by compound 10 in A549 and NCI-H1299 cells are described.

ne of the hallmarks of human cancer is the ability to acquire chemotherapy resistance by evading apoptosis.1 However, most clinical anticancer agents induce cancer cell death via caspase-dependent apoptosis.2 Therapies based on the induction of nonapoptotic cell death, such as paraptosis, autophagy, oncosis, methuosis, and necroptosis, would be an alternative way to treat cancers with apoptosis resistance.3−6 Paraptosis, a nonapoptotic form of programmed cell death, is characterized by extensive cytoplasmic vacuolation derived from swelling of the endoplasmic reticulum (ER) and/or mitochondria.7 While the detailed mechanisms are not fully understood, paraptosis has been found to be an effective method for killing apoptosis-resistant cancers. Importantly, natural products have been demonstrated to be a valuable source of paraptosis inducers.8−10 Liverworts are rich in bioactive substances, including aromatic compounds and terpenoids.11−15 Radula species, a small stemleafy liverwort, are widely distributed and have been investigated extensively.16,17 Recent studies have shown that Radula liverwort species are abundant in prenylated bibenzyls,11,18 which show superior cytotoxic activity.19 The bioactive chemical constituents of the adnascent Chinese liverwort Radula constricta Steph. were investigated.20,21 Consequently, nine new bibenzyl derivatives, radstrictins A−I (1−9), were isolated, and six of them were obtained as a racemate or scalemic mixtures [(±)-radstrictins A−F (1−6)]. In addition, 11 known compounds (10−20) were also isolated with compound 10 showing marked suppression of cell viability in tested cancer cell © XXXX American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION Structure Elucidation. (±)-Radstrictin A (1), a yellowish oil, was assigned the molecular formula C21H24O5 on the basis of the protonated molecule detected at m/z of 357.1698 [M + H]+ (calcd 357.1697) as showed by (+)-HRESIMS and 1D NMR data (Tables 1 and 2), which is consistent with 10 indices of hydrogen deficiency. The IR spectrum showed an absorption at 3244 cm−1 due to a hydroxy group as well as absorptions at 1647, 1622, 1579, and 1495 cm−1 that were attributed to aromatic moieties. The 1H NMR data (Table 1) displayed signals for a monosubstituted and a pentasubstituted aromatic ring [δH 7.31 (m, 2H), 7.20 (m, 2H), 7.22 (m, 1H), and 6.39 (s, 1H)]. In addition, a phenolic (δH 12.11, s), an olefinic methylene group (δH 4.88, s and 5.01, s), an oxygenated methine [δH 4.37 (t, J = 5.7 Hz)], and two methyl singlets (δH 1.86, s and 3.95, s) including a methoxy group were observed. The terminal double Received: October 25, 2018

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DOI: 10.1021/acs.jnatprod.8b00897 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

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Chart 1

Table 1. 1H NMR Spectroscopic Data for Compounds 1−7 in CDCl3 1b

2b

3a

4b

5a

6b

7b

position

δH, mult. (J in Hz)







1a 1b 2 4a 4b 5a 5b 6 7 9 10 1′ 5′ 8′ 1″ 2″ 4″, 8″ 5″, 7″ 6″ HO-2′

3.18, dd (14.8, 5.7) 2.82, m 4.37, t (5.7) 5.01, s 4.88, s 1.86, s

3.13, m 3.07, dd (15.5, 8.6) 4.71, t (8.6) 1.22, s

2.93, dd (17.3, 5.2) 2.72, dd (17.3, 5.2) 3.85, t (5.2) 1.38, s

2.88, dd (16.9, 5.2) 2.66, dd (16.9 5.2) 3.82, t (5.2) 1.33, s

2.62, t (7.4) 1.64, t (7.4) 1.23, s

2.73, dd (16.5, 5.3) 2.49, dd (16.5, 5.3) 3.80, t (5.3) 1.28, s

1.34, s

1.33, s

1.31, s

1.55, td (6.9, 2.9)

1.54, m

2.04, m 5.12, t (7.4) 1.61, s 1.68, s 6.28, d (2.1) 6.27, d (2.1)

2.09, m 5.08, t (6.9) 1.59, s 1.67, s 6.22, d (2.5) 6.33, d (2.5)

2.79, dd (15.3, 8.9) 2.75, dd (15.3, 8.9) 4.23, d (8.9) 5.16, s 4.94, s 2.20, m 2.10, m 2.19, m 5.11, m 1.60, s 1.69, s 6.35, d (2.6) 6.32, d (2.6)

2.82, m 2.83, m 7.17, m 7.28, m 7.19, m

2.78, m 2.85, m 7.16, m 7.29, m 7.20, m

2.81, m 2.83, m 7.16, m 7.28, m 7.20, m

a1

6.39, s 3.95, s 3.13, m 2.83, m 7.20, m 7.31, m 7.22, m 12.11, s

6.27, s 3.95, s 3.17, m 2.82, m 7.20, m 7.31, m 7.22, m 11.78, s

H NMR was recorded at 400 MHz.

6.30, s 3.96, s 3.13, m 2.83, m 7.20, m 7.31, m 7.22, m 12.07, s b1

6.18, s 3.88, s 2.77, m 2.83, m 7.17, m 7.26, m 7.19, m

H NMR was recorded at 600 MHz.

A bibenzyl skeleton22,23 was proposed based on the aromatic moieties, in combination with the −CH2−CH2− fragment (δH 3.13 and 2.83) suggested by a 1H−1H COSY experiment and confirmed by the HMBC signals from H2-1″ to C-3′ (δC 104.1), C-5′ (δC 112.1), and C-3″ (δC 145.1) and from H2-2″ to C-4′

bond (δC 146.9 and 110.3) was confirmed by the 13C NMR (Table 2) and HSQC data. Three methylenes (δC 38.9, 38.3, and 29.0), an oxygenated secondary carbon (δC 77.6), 12 aromatic carbons (δC 163.1−104.1), and an ester carbonyl carbon (δC 172.4) carrying a methoxy (δC 52.0) group were also observed. B



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Table 2. 13C NMR Spectroscopic Data for Compounds 1−7 in CDCl3

a13

position

1b

2b

3a

4b

5a

6b

7b

1 2 3 4 5 6 7 8 9 10 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 1″ 2″ 3″ 4″, 8″ 5″, 7″ 6″

29.0 77.6 146.9 110.3 18.5

27.7 91.2 72.0 23.7 25.9

26.0 69.1 77.7 22.1 24.7

26.1 69.0 77.3 21.8 24.5

111.0 163.1 104.1 142.1 112.1 161.1 172.4 52.0 38.9 38.3 145.1 128.3 128.4 125.9

111.5 164.7 105.1 141.9 105.0 160.2 171.9 52.0 39.3 38.5 147.5 128.3 128.4 126.0

105.1 163.2 103.9 142.1 111.8 157.5 172.2 52.0 38.9 38.4 144.3 128.2 128.4 125.9

104.7 151.5 115.9 141.6 107.7 155.0 168.8 52.0 35.7 37.6 139.6 128.4 128.4 126.0

18.9 41.3 74.1 26.5 41.7 22.9 123.9 132.5 17.7 25.7 101.7 155.3 119.8 141.5 108.1 155.4

28.3 68.3 78.1 19.1 36.9 21.7 124.0 132.0 17.6 25.7 102.0 153.7 109.6 142.5 108.7 154.8

32.6 78.6 151.2 110.1 31.6 26.6 123.6 132.4 17.7 25.7 102.8 157.2 117.1 142.3 108.7 154.9

35.4 37.7 141.8 128.3 128.4 126.0

34.5 36.6 141.5 128.4 128.5 126.0

35.7 37.7 141.5 128.3 128.4 126.1

C NMR was recorded at 100 MHz.

b13

C NMR was recorded at 150 MHz.

Figure 1. Key 1H−1H COSY and HMBC correlations of compounds 1−9.

(δC 142.1) and C-4″/8″ (δC 128.3). The remaining five carbons were speculated to be an isopentenyl moiety according to the HMBC signals from H2-1 (δH 2.82 and 3.18) to C-2 (δC 77.6) and C-3 (δC 146.9) and from H2-4 to C-2, C-3, and C-5 (δC 18.5), along with the spin system C-1(H2)−C-2(H) observed as

cross-peaks in the 1H−1H COSY spectrum. Therefore, 1 was determined to be a prenylated bibenzyl. The HMBC signals from H2-1 to C-1′ (δC 111.0), C-2′ (δC 163.1), and C-6′ (δC 161.1) verified the connectivity between the bibenzyl and the isopentenyl moieties via C-1 and C-1′. The ester group at C-3′ C



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inferred as shown in Figure 1. Compound 3 was also found to be a scalemic mixture using the methodology described for 1. Chiral-phase resolution and comparison of experimental and computed ECD data permitted the assignment of the absolute configurations of (−)-3a and (+)-3b as 2S and 2R, respectively (Figures S29, S30, Supporting Information). (±)-Radstrictin D (4), a transparent oil, was assigned the same molecular formula, C21H24O5, as 3, based on its HRESIMS [M + H]+ ion at m/z 357.1702 and 13C NMR data. The structure of 4 was deduced by comparing its spectroscopic data with those of 3. In contrast to compound 3, the signal for the hydroxy group was absent in the 1H NMR spectrum of compound 4, and there was an upfield shift of the carbon signal at C-2′ (δC 151.5); thus, the hydroxy group was likely located at C-6′. This conclusion was confirmed by the HMBC signals from H2-1 (δH 2.88 and 2.66) to C-2′ and C-6′ and from H-2 (δH 3.82) to C-1′ (δC 104.7). The specific rotation value of 4 approached zero, suggesting that 4 was also a scalemic mixture. Since the experimental and calculated ECD curves did not match well, the OR values of 4a and 4b were calculated and the values of +24.3 and −24.3 were in agreement with the experimental values of +23.7 and −23.1, respectively. Therefore, the absolute configurations of (+)-4a and (−)-4b were assigned as 2R and 2S, respectively (Figures S39, S40, Supporting Information). (±)-Radstrictin E (5), a colorless oil, had the molecular formula C24H32O3, with nine indices of hydrogen deficiency according to the HRESIMS (m/z 369.2427 [M + H]+, calcd 369.2424) and NMR data. The 1H NMR data showed signals for a monosubstituted and a tetrasubstituted aromatic ring [δH 7.28 (m, 2H), 7.17 (m, 2H), 7.19 (m, 1H), 6.27 (d, J = 2.1 Hz, 1H), 6.28 (d, J = 2.1 Hz, 1H)]. The 1D NMR and HSQC data showed three methyl groups (δC 26.5, 25.7, and 17.7), six methylene groups (δC 18.9, 41.3, 41.7, 22.9, 35.4, and 37.7), an oxygenated tertiary carbon (δC 74.1), and a double bond (δC 123.9 and 132.5). As 14 of the carbons can be attributed to a bibenzyl unit, the remaining 10 carbons were speculated to comprise a geranyl group, as illustrated in the structure depicted for 5. Comparison of the NMR data of compound 5 with those of 3,5-dihydroxy-2geranylbibenzyl (16)17 revealed that their structures were similar except for the Δ2(3) double bond in 16 being hydrated to manifest in a methylene group at C-2 (δC 41.3) and an oxygenated tertiary C-3 (δC 74.1) in 5. The methylene group was supported by the HMBC signals from H2-2 to C-1 (δC 18.9), C-3 (δC 74.1), and C-3′ (δC 119.8), along with the spin system C-1(H2)−C-2(H2) observed in the 1H−1H COSY spectrum. The oxygenated tertiary carbon was inferred via the HMBC signals from H3-4 to C-2, C-3, and C-5 (δC 41.7). Compound 5 was also a scalemic mixture, as evidenced by the two peaks not in a 1:1 ratio that were observed on a chiral-phase column. The experimental and calculated ECD data defined the absolute configurations of (+)-5a and (−)-5b as 3R and 3S, respectively (Figures S49, S50, Supporting Information). (±)-Radstrictin F (6) had a molecular formula of C24H30O3, based on its HRESIMS (m/z 367.2274 [M + H]+, calcd 367.2268) and NMR data (Tables 1 and 2), indicative of 10 indices of hydrogen deficiency. Comparison of the NMR data of compound 6 with those of the known compound 2(S)-7hydroxy-2-methyl-2-(4-methyl-3-pentenyl)-5-(2-phenylethyl)chromene (o-cannabichromene) (15)22 revealed that the compounds shared the same bibenzyl unit; however, the Δ1(2) double bond of the monoterpenoid side-chain in 15 was hydrated to form a methylene (δH 2.49 and 2.73) and an

was supported by the HMBC signals from H2-1″ to C-3′ and from H-5′ to C-3′. Using CDCl3 as the solvent, an exchangeable hydroxy proton resonated at δH 12.11, a chemical shift indicating hydrogen bond formation with the carbonyl group. Thus, the hydroxy groups were located at C-2′ and C-6′, based on the HMBC signals from H2-1 to C-2′ and C-6′ and from HO-2′ to C-1′, C-2′, and C-3′. Consequently, the 2D structure of 1 was deduced as depicted (Figure 1) and closely resembled the structure of methyl 2,4-dihydroxy-3-(3-methyl-2-butenyl)-6phenethylbenzoate (10).24 Analysis of the data revealed a Δ3(4) double bond in 1 instead of a Δ2(3) double bond in 10 as well as the presence of a C-2 hydroxy group. A zero specific rotation value suggested that compound 1 was likely a racemate. This prediction was confirmed by the presence of two peaks, which were obtained on a chiral-phase column in a 1:1 ratio. The enantiomers (+)-1a and (−)-1b were obtained by chiral-phase separation; however, their experimental electronic circular dichroism (ECD) curves displayed weak Cotton effects. Since the specific rotation values of (+)-1a or (−)-1b are determined by the single C-2 stereogenic center, their absolute configurations could be defined via optical rotation (OR) calculations. The computed values for the R and S configurations were +31.4 and −31.4, which were consistent with the experimental values of (+)-1a (+30.8) and (−)-1b (−29.8), respectively. Thus, the structures of (+)-1a and (−)-1b were defined as shown in the chart (Figures S9, S10, Supporting Information). (±)-Radstrictin B (2), a colorless oil, was assigned the same molecular formula, C21H24O5, as compound 1, based on its HRESIMS (m/z 357.1701 [M + H]+, calcd 357.1697) and NMR data (Tables 1 and 2). The 1D NMR data of 2 resembled those of 1, indicating a prenylated bibenzyl derivative. However, the olefinic C-3 methylene group in 1 was replaced by a methyl group (δH 1.22) in 2, as revealed by the 1H NMR spectrum. The gem-dimethyl moiety (δC 23.7 and 25.9) and an oxygenated secondary carbon (δC 72.0) were established by the 13C NMR and HSQC spectra, along with the HMBC signal from H3-4 to C-2 (δC 91.2), C-3, and C-5. The presence of a dihydrobenzofuran moiety was inferred from the remaining index of hydrogen deficiency and by the downfield chemical shift of C-2 compared to 1. The connectivity between the bibenzyl and isopentenyl moieties via C-1 and C-1′ as well as the presence of an ether linkage between C-2 and C-6′ were determined by the observation of a phenolic group at C-2′ [HO-2′ (δH 11.78)] and were further corroborated by the HMBC signals from HO2′ to C-1′ (δC 111.5), C-2′ (δC 164.7), and C-3′ (δC 105.1) and from H2-1 (δH 3.13 and 3.07) to C-1′, C-2′, and C-6′ (δC 160.2). The [α]20D value of +1.1 was indicative of a scalemic mixture. The two enantiomers of 2 displayed typical antipodal Cotton effects by comparison of their experimental and calculated ECD data, thus allowing the absolute configurations of (+)-2a and (−)-2b to be defined as 2R and 2S, respectively (Figures S19, S20, Supporting Information). (±)-Radstrictin C (3), an amorphous powder, possessed the same molecular formula, C21H24O5, as 2, affirmed by the (+)-HRESIMS ion at m/z 357.1699 [M + H]+. The presence of a chromane moiety in 3 rather than the dihydrobenzofuran unit in 2 was supported by the upfield shift of H-2 (δH 3.85) in the 1H NMR data and by the chemical shift of C-2 (δC 69.1) in the 13C NMR data. The downfield shift of C-3 (δC 77.7), combined with the HMBC signals from H2-1 (δH 2.93 and 2.72) to C-3, C-2′ (δC 163.2), and C-6′ (δC 157.5) and from HO-2′ (δH 12.07) to C-1′ (δC 105.1) and C-2′, led to the determination of an ether linkage between C-3 and C-6′. Thus, its 2D structure was D



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Table 3. 1H and 13C NMR Spectroscopic Data for Compounds 8 in CDCl3 and 9 in Methanol-d4

oxygenated methine (δH 3.80) in 5, which was supported by the downfield chemical shift of C-2 (δC 68.3) and the HMBC signals from H-2 to C-1 (δC 28.3) and C-3 (δC 78.1), as well as by 1 H−1H COSY coupling in the spin system C-1(H2)−C-2(H). NOESY data suggested that the C-2 hydroxy group had the same orientation as the C-4 methyl, as indicated by the signals from H2 to H2-5 and H2-6 (Figure 2). Compound 6 was determined to

8b position 1 2 3 4 5 1′ 2′ 3′ 4′ 5′ 6′ 1″ 2″ 3″ 4″ 5″ 6″ 7″

Figure 2. Key NOESY correlations of compound 6.

8″ HO-2′ a13

δH, mult. (J in Hz) 6.28, s 4.55, s 2.07, d (1.1)

6.40, d (1.6) 6.60, d (1.6) 2.84, m 2.91, m

6.74, m 7.09, m 6.86, ddd (7.4, 7.3, 0.8) 7.08, m 13.40, s

9a δC 193.2 132.0 154.4 72.4 22.9 112.3 164.9 110.7 152.4 113.0 160.5 36.2 31.1 127.3 153.4 115.2 127.5 120.9 130.3

C NMR was recorded at 100 MHz. 150 MHz.

be a scalemic mixture, and by using the same resolution method described above, (+)-6a was assigned the (2S, 3R) absolute configuration and (−)-6b the (2R, 3S) absolute configuration (Figures S60, S61, Supporting Information). Radstrictin G (7), a colorless oil, was assigned the same molecular formula as 6, C24H30O3, based on its HRESIMS (m/z 367.2271 [M + H]+) and NMR data. The 1D NMR data (Tables 1 and 2) were similar to those of 6. The major differences involved the cleavage of the ether linkage and the substitution of the methyl group at C-3 by a terminal vinyl group (δH 5.16 and 4.94) in 7. The HMBC signals from H2-4 to C-2 (δC 78.6), C-3 (δC 151.2), and C-5 (δC 31.6) confirmed the position of the double bond. Additionally, compounds 6 and 7 possessed the same indices of hydrogen deficiency, while the presence of a Δ3(4) double bond in 7 indicated the cleavage of the ether linkage. The specific rotation value, [α]20D, of +22.1 (c 0.1, MeOH) was indicative of an optically pure compound, supported by the observation of only one peak using a chiralphase column. Based on the coincidence of the calculated and experimental ECD data, the absolute configuration of 7 was determined as 2R (Figures S70, S71, Supporting Information). Radstrictin H (8), a yellowish oil, had the molecular formula C19H18O4, based on HRESIMS [M + H]+ m/z 311.1278 (calcd 311.1278) and 13C NMR data, which indicated 11 indices of hydrogen deficiency. Signals in the 1H NMR spectrum [δH 6.74 (d, J = 7.4 Hz, 1H), 7.09 (m, 1H), 6.86 (td, J = 7.4, 0.8 Hz, 1H), 7.08 (m, 1H), 6.40 (d, J = 1.6 Hz, 1H), and 6.60 (d, J = 1.6 Hz, 1H)] demonstrated the presence of a disubstituted and a tetrasubstituted aromatic ring in 8. The 13C NMR (Table 3) and HSQC data for 8 revealed the presence of an oxymethylene group (δC 72.4), two olefinic carbons (δC 132.0 and 154.4), and a ketocarbonyl carbon (δC 193.2). Except for the presence of a hydroxy group at C-4″ (δC 153.4), which was confirmed by the HMBC signal from H2-2″ (δH 2.91) to C-4″, the 1D NMR data of 8 were similar to those of 6-hydroxy-3-methyl-8phenethylbenzo[b]oxepin-5(2H)-one,25 which was previously isolated from the epiphytic liverwort Marsupidium epiphytum.

δH, mult. (J in Hz) 7.75, d (12.3) 6.18, d (12.3) 4.42, s

6.48, d (1.2) 6.43, d (1.2) 2.75, m 2.82, m

6.71, m 6.96, m 6.66, ddd (7.4, 7.3, 0.8) 6.93, m b13

δC 138.8 126.5 199.6 78.8 115.4 159.7 111.9 150.2 111.7 161.6 37.4 33.1 128.9 156.4 115.8 128.2 120.4 131.1

C NMR was recorded at

Radstrictin I (9), yellowish needles, had a molecular formula of C18H16O4 as defined by analysis of the NMR data and the presence of an [M + H]+ ion at m/z 297.1122 (calcd 297.1121) in the HRESIMS spectrum. The mass of 9 was 14 Da lower than that of 8, indicating that 9 is a demethyl derivative of 8. Comparison of the 1D NMR data (Table 3) of 8 and 9 suggested that the carbonyl group was present at C-3 (δC 199.6) in 9, as supported by the HMBC signal from H-1 (δH 7.75) to C-3. Additionally, the HMBC signals from H2-4 (δH 4.42) to C-2 (δC 126.5) and C-3 along with the 1H−1H COSY spin system C1(H)−C-2(H) verified the presence of a Δ1(2) double bond in 9. Single-crystal X-ray diffraction analysis confirmed the structure of 9 (Figure 3, CCDC 1858356). The following known compounds were also isolated, as determined by comparison of their experimental and reported spectroscopic data: methyl 2,4-dihydroxy-3-(3-methyl-2-butenyl)-6-phenethylbenzoate (10),24 radulanin A (11),17 radulanin L (12),17 7-hydroxy-2,2-dimethyl-5-(2-phenylethyl)chroman

Figure 3. ORTEP drawing of compound 9. E



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(13),17 7-methoxy-2,2-dimethyl-5-(2-phenylethyl)chroman (14),26 2(S)-7-hydroxy-2-methyl-2-(4-methyl-3-pentenyl)-5(2-phenylethyl)chromene (o-cannabichromene) (15),22 3,5dihydroxy-2-geranylbibenzyl (16),17 aglaiabbrevin C (17),27 bibenzyl/o-cannabicyclol hybrid (18),19 rasumatranin B (19),19 and rasumatranin A (20).19 Exemplified by the skeletons of these prenylated bibenzyls, their biosynthetic pathways were postulated (Scheme S1, Supporting Information). Starting from 3,5-dihydroxybibenzyl (Ι), coupling with dimethylallyl diphosphate (DMAPP) formed the key precursor 3,5-dihydroxy-4-(3-methyl-2-butenyl)bibenzyl (II).17 Subsequent dehydration, hydration, cyclization, oxidation, and reduction processes produced compounds 1−4 and 8−12, while compounds 13 and 14 were formed from the precursor 3,5-dihydroxy-2-[3-methyl-(Z)-butenyl]bibenzyl (IV).17 Similarly, the 3,5-dihydroxybibenzyl (I) and geranyl diphoshate (GPP) could be combined through an analogous series of reactions to give compounds 5−7 and 15−20 from precursor 16. Compound 10 Inhibits Cancer Cell Proliferation. The compounds were evaluated for their cytotoxic activity against the NCI-H1299, A549, HepG-2, SMMC-7721, MCF-7, U251, Sw620, HT29, and KB cell lines with adriamycin as a positive control (Table 4). Among these compounds, 10, 12, 13, 17, and

18 induced cytoplasmic vacuolization in A549 cells (Figure S88, Supporting Information). Compound 10 was noteworthy because it significantly reduced cell viability in all the tested cancer cell lines and induced a quick and striking accumulation of cytoplasmic vacuolization within 3 h (Figure 4A,B). Therefore, 10 was chosen for further study to explore its potential cytotoxic mechanism. Compound 10 Induces Nonapoptotic Cell Death in Cancer Cells. As 10 induced vacuolization in the cytoplasm, we hypothesized that 10 induced cell death via a nonapoptotic process. To validate this hypothesis, annexin V/propidium iodide (PI) staining was used to investigate whether the toxicity of 10 was related to apoptosis. The flow cytometry results showed that no significant changes in the percentages of annexin V-positive cells (6.6% in A549 and 4.1% in NCI-H1299) were observed after treatment of the cells with 10 compared with the control cells (4.6% in A549 and 4.5% in NCI-H1299) (Figure 5A and Figure S89, Supporting Information). The data in Figure 5D demonstrated that PARP cleavage was not detected after treatment with 10. After pretreating the cells with the pancaspase inhibitor Z-VAD-FMK for 1 h, cell viability and vacuolation did not noticeably change (Figure 5B,C). These results clearly indicated that compound 10 caused a nonapoptotic process leading to cell death. Compound 10 Induces Cell Death through Mitochondria-Derived Paraptosis. Following treatment of cancer cells with compound 10, many vacuoles were observed in the cytosol and at the perinuclear region. The result in Figure 6A showed that untreated cells displayed a filamentous and elongated morphology upon Mito-Tracker staining, numerous fluorescent vacuoles were observed in the cells at 3 h after treatment with 10, and fusion among swollen mitochondria progressed further from 6 to 24 h. The above results suggested that the vacuoles originated from the swollen mitochondria. Furthermore, the fluorescence intensity during flow cytometry notably decreased in cells treated with 10 (Figure 6B). In contrast to apoptosis, in which HMGB1 was localized to the nucleus, HMGB1 was released from the nucleus during paraptosis.28 As shown in Figure 6E, HMGB1 was translocated from the nucleus to the cytoplasm. The above results indicated that compound 10 induces cell death through paraptosis. To test whether compound 10 induces mitochondria dysfunction, the mitochondrial membrane potential (MMP, ΔΨm) was investigated with JC-1 dye by flow cytometry. The results suggested that 10 caused a loss of ΔΨm in the cells. After exposure to 10 for 1 h, the ΔΨm were reduced in A549 and NCI-H1299 cells to 50%

Table 4. Cytotoxicity of Compounds 10, 18, and Adriamycin in Several Cancer Cell Lines IC50 (μM)b cell linea

compound 10

compound 18

adriamycin

NCI-H1299 A549 HepG-2 SMMC7721 MCF-7 U251 Sw620 HT29 KB

5.1 ± 0.3 6.0 ± 0.1 6.3 ± 0.2 8.6 ± 1.3 − 9.0 ± 0.2 6.3 ± 1.4 5.0 ± 0.5 6.7 ± 1.6

7.5 ± 0.5 9.8 ± 0.2 −c − − − − − −

0.8 ± 0.4 1.8 ± 0.3 1.5 ± 1.5 0.8 ± 1.6 1.0 ± 0.9 2.9 ± 1.6 1.2 ± 0.4 3.8 ± 0.7 3.2 ± 0.3

a

Cells were treated with compounds 10, 18 and adriamycin for 48 h, and viability values were determined by MTT assays. Results are expressed as the mean IC50 of three experiments. bOther inactive compounds (IC 50 > 10 μM) are shown in the Supporting Information. cNot active (IC50 > 10 μM). Data are mean ± SD (n = 3).

Figure 4. Compound 10 reduced the survival rate and induced extensive vacuolization. (A) Cytotoxicity of 10 on A549 and NCI-H1299 cells. Cells were treated with 10 for 48 h. Cell viability rate, which is denoted as a percentage of untreated control (compound 10, 0 μM) was estimated by the MTT assay. Data are mean ± SD (n = 3). *P < 0.05 and **P < 0.01, vs control. (B) A549 and NCI-H1299 cells were treated with 10 at a concentration of 10 μM, and the morphological changes were observed by optical microscopy at the indicated time. Bar = 100 μm. F



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Figure 5. Compound 10 induced a nonapoptotic process in A549 and NCI-H1299 cells. (A) Representative apoptotic profile of A549 and NCI-H1299 cells treated or untreated with 10 from flow cytometric analysis. (B) Optical microscopy images of A549 and NCI-H1299 cells treated with DMSO (control), 20 μM Z-VAD-FMK, 10 μM 10, or their combinations for 3 h. Bar = 100 nm. (C) A549 and NCI-H1299 cells were treated with DMSO (control), 20 μM Z-VAD-FMK, 5 μM 10, or their combinations for 24 h. Viability values were determined by MTT assays. Data are mean ± SD (n = 3). (D) Western blot analysis of the indicated proteins after A549 and NCI-H1299 cells were treated with different concentrations of 10 for 24 h.

mitochondria via increasing ROS levels, thereby decreasing the mitochondrial membrane potential. Thus, the identification of prenylated bibenzyls from liverworts that could induce nonapoptotic cell death in cancer cells provides an important strategy for discovering novel cytotoxic compounds.

and 25% of the level of the 0 h control, respectively (Figure 6C). Mitochondrial damage and reactive oxygen species (ROS) elevation have been reported to be closely related events.29,30 2′,7′-Dichlorodihydrofluorescein diacetate (DCFH-DA) was used to detect ROS levels by flow cytometry. The results indicated that 10 caused a dramatic increase in ROS levels in a time-dependent manner in A549 and NCI-H1299 cells (Figure 6D). Comparing those results with the control cells, the mean fluorescence intensity of DCF increased approximately 20-fold in A549 cells and 30-fold in NCI-H1299 cells after treatment with 10 for 1 h. Overall, nine new prenylated bibenzyl derivatives [radstrictins A−I (1−9)], six of which were isolated as a racemate or scalemic mixtures [(±)-radstrictins A−F (1−6)], and 11 known compounds have been obtained from R. constricta Steph. Among the compounds tested for cytotoxicity, compound 10 showed lower IC50 values in cytotoxicity tests as well as cell vacuolization in A549 and NCI-H1299 cells. Additional experiments indicated that 10 induces mitochondria-derived paraptosis in cancer cells and disrupts the functions of

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EXPERIMENTAL SECTION

General Experimental Procedures. An X-6 micromelting point apparatus was used to measure the melting points. Optical rotations were recorded using an MCP 200 polarimeter (Anton Paar) at 589 nm and calculated in the Gaussian 09 program package. A Shimadzu UV2450 spectrophotometer was used to record UV data. A Chirascan spectropolarimeter was used to measure the ECD spectra. A Nicolet iN 10 micro FTIR spectrometer was employed to record IR spectra. Bruker Avance DRX-600 and Bruker AV 400 spectrometers were used to acquire NMR spectra, using methanol-d4 or CDCl3 as the solvent, with tetramethylsilane as the internal standard. An LTQ-Orbitrap XL instrument was used to obtain the HRESIMS spectra. HPLC was carried out on a Shimadzu HPLC system consisting of an LC-20AT pump with an SPD-M20A detector, a DGU-20A5R degasser, and a Shim-pack GIST-C18 5 μm column (10 × 250 mm). Preliminary HPLC G



Article

Figure 6. Compound 10 induced cell death via mitochondria-derived paraptosis. (A, B) A549 and NCI-H1299 cells were incubated with 10 at indicated time points and analyzed using the Mito-Tracker staining assay. Images were obtained by fluorescence microscopy. Bar = 10 μm. Furthermore, intensity of mitochondria fluorescence from 10 000 cells per sample was measured by flow cytometry. Cells with decreased red fluorescence (“pale” cells) were gated, and their percentages are indicated. (C) Compound 10 induced the loss of ΔΨm. After cells were treated with 10 for indicated time points, ΔΨm was measured by flow cytometry. (D) A549 and NCI-H1299 were incubated with 10 at indicated time points and then stained with DCFH-DA. ROS was measured by flow cytometry. Data are mean ± SD (n = 3). *P < 0.05 and **P < 0.01 vs control. (E) Immunofluorescence staining of HMGB1 (red) in cells. Nuclei were counterstained with DAPI (blue). A549 and NCI-H1299 cells were treated with 10 μM compound 10 for 24 h. Bar = 10 μm. H



Article

(±)-Radstrictin A (1): yellowish oil (MeOH); [α]20D 0.0 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 268 (3.63) nm, λmax (log ε) 305 (3.27) nm; IR νmax 3244, 2950, 1647, 1622, 1579, 1495 cm−1; 1H NMR (CDCl3, 600 MHz), see Table 1, and 13C NMR (CDCl3, 150 MHz), see Table 2; HRESIMS m/z 357.1698 [M + H]+ (calcd 357.1697). (+)-Radstrictin A (1a): [α]20D +30.8 (c 0.1, MeOH); ECD (c 0.5, MeOH) λmax (Δε) 207 (+15.37), 218 (+6.25), 223 (+7.18), 233 (−1.66) nm. (-)-Radstrictin A (1b): [α]20D −29.8 (c 0.1, MeOH); ECD (c 0.5, MeOH) λmax (Δε) 205 (+4.31), 219 (+9.31), 225 (+0.67), 228 (+1.25) nm. (±)-Radstrictin B (2): colorless oil (MeOH); [α]20D +1.1 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 275 (2.81) nm; IR νmax 3560, 3475, 2924, 1656 cm−1; 1H NMR (CDCl3, 600 MHz), see Table 1, and 13C NMR (CDCl3, 150 MHz), see Table 2; HRESIMS m/z 357.1701 [M + H]+ (calcd 357.1697). (+)-Radstrictin B (2a): [α]20D +31.0 (c 0.1, MeOH); ECD (c 1.0, MeOH) λmax (Δε) 205 (−13.83), 270 (+1.17) nm. (−)-Radstrictin B (2b): [α]20D −29.8 (c 0.1, MeOH); ECD (c 1.0, MeOH) λmax (Δε) 209 (+10.52), 271 (−1.21) nm. (±)-Radstrictin C (3): amorphous powder (MeOH); [α]20D −0.9 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 270 (2.71) nm, λmax (log ε) 305 (2.29) nm; IR νmax 3386, 2969, 1641, 1432 cm−1; 1H NMR (CDCl3, 400 MHz), see Table 1, and 13C NMR (CDCl3, 100 MHz), see Table 2; HRESIMS m/z 357.1699 [M + H]+ (calcd 357.1697). (−)-Radstrictin C (3a): [α]20D −23.5 (c 0.1, MeOH); ECD (c 0.5, MeOH) λmax (Δε) 208 (−4.92) nm. (+)-Radstrictin C (3b): [α]20D +24.1 (c 0.1, MeOH); ECD (c 0.5, MeOH) λmax (Δε) 209 (+4.84) nm. (±)-Radstrictin D (4): transparent oil (MeOH); [α]20D +1.3 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 282 (1.93) nm; IR νmax 3331, 2963, 1699 cm−1; 1H NMR (CDCl3, 600 MHz), see Table 1, and 13C NMR (CDCl3, 150 MHz), see Table 2; HRESIMS m/z 357.1702 [M + H]+ (calcd 357.1697). (+)-Radstrictin D (4a): [α]20D +23.7 (c 0.1, MeOH); ECD (c 0.1, MeOH) λmax (Δε) 207 (−3.12), 214 (+0.65), 207 (−1.73) nm. (−)-Radstrictin D (4b): [α]20D −23.1 (c 0.1, MeOH); ECD (c 0.2, MeOH) λmax (Δε) 205 (+3.24), 215 (−0.69), 205 (+0.74) nm. (±)-Radstrictin E (5): colorless oil (MeOH); [α]20D −0.8 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 278 (1.78) nm; IR νmax 3342, 2925, 1723, 1602, 1453 cm−1; 1H NMR (CDCl3, 400 MHz), see Table 1, and 13 C NMR (CDCl3, 100 MHz), see Table 2; HRESIMS m/z 369.2427 [M + H]+ (calcd 369.2424). (+)-Radstrictin E (5a): [α]20D +25.3 (c 0.1, MeOH); ECD (c 0.2, MeOH) λmax (Δε) 207 (+3.61) nm. (−)-Radstrictin E (5b): [α]20D −26.0 (c 0.1, MeOH); ECD (c 0.2, MeOH) λmax (Δε) 206 (−3.33) nm. (±)-Radstrictin F (6): colorless oil (MeOH); [α]20D −1.3 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 277 (2.31) nm; IR νmax 3342, 2926, 1615, 1451 cm−1; 1H NMR (CDCl3, 600 MHz), see Table 1, and 13C NMR (CDCl3, 150 MHz), see Table 2; HRESIMS m/z 367.2274 [M + H]+ (calcd 367.2268). (+)-Radstrictin F (6a): [α]20D +31.8 (c 0.1, MeOH); ECD (c 0.4, MeOH) λmax (Δε) 205 (−4.11) nm. (−)-Radstrictin F (6b): [α]20D −33.0 (c 0.1, MeOH); ECD (c 0.4, MeOH) λmax (Δε) 205 (+3.96) nm. Radstrictin G (7): colorless oil (MeOH); [α]20D +22.1 (c 0.1, MeOH); colorless oil (MeOH); ECD (c 0.3, MeOH) λmax (Δε) 205 (+4.16) nm; UV (MeOH) λmax (log ε) 284 (2.58) nm; IR νmax 3345, 2966, 1602, 1138 cm−1; 1H NMR (CDCl3, 600 MHz), see Table 1, and 13 C NMR (CDCl3, 150 MHz), see Table 2; HRESIMS m/z 367.2271 [M + H]+ (calcd 367.2268). Radstrictin H (8): yellowish oil (MeOH); UV (MeOH) λmax (log ε) 256 (3.46) nm, λmax (log ε) 301 (3.58) nm; IR νmax 3397, 2929, 1628, 1208 cm−1; 1H (CDCl3, 600 MHz) and 13C NMR (CDCl3, 150 MHz), see Table 3; HRESIMS m/z 311.1278 [M + H]+ (calcd 311.1278). Radstrictin I (9): yellowish crystals (MeOH); mp 137−138 °C; UV (MeOH) λmax (log ε) 269 (3.68) nm, λmax (log ε) 305 (3.36) nm; IR νmax 3446, 2921, 1603, 1081 cm−1; 1H (CD4O, 400 MHz) and 13C

analyses of the racemate and scalemic mixtures were performed on a Waters Delta 600 system with a CHIRALPAK AD-H 5 μm column (4.6 × 250 mm) and a Waters 996 photodiode array detector. All reagents used during the experiments were of analytical grade. Column chromatography used a conventional packing, such as reversed-phase C18 (40−63 μm, FuJi), silica gel (200−300 mesh; Qingdao Haiyang Chemical Co. Ltd., Qingdao, P. R. China), and Sephadex LH-20 (25− 100 μm; Pharmacia Biotek, Denmark). Thin-layer chromatography was implemented with silica gel GF254 plates (Qingdao Haiyang Chemical Co. Ltd.). Dark spots of compounds were observed under a threepurpose ultraviolet spectrometer at 254 and 365 nm. Compounds were colored by spraying with H2SO4−EtOH (1:9, v/v) followed by heating. Crystal structure analysis was completed by X-ray diffraction with Cu Kα radiation. Plant Material. The sample of liverwort Radula constricta was identified by Dr. Jinchuan Zhou, School of Pharmacy, Linyi University, People’s Republic of China, derived from the Yunwu Temple, Shaanxi Province, People’s Republic of China, in July 2017. The sample (No. 20170723-009) has been placed at the Department of Natural Products Chemistry, School of Pharmaceutical Sciences, Shandong University, People’s Republic of China. Extraction and Isolation. Crushed and air-dried plant material of R. constricta (70.1 g) was refluxed with 95% EtOH. The crude extract (8.7 g) was initially separated via MCI gel column chromatography using a gradient elution with a solvent of H2O−MeOH (7:3 to 0:1) to give fractions 1−6. Fraction 2 (1.3 g) was further purified on a silica gel column [acetone−petroleum ether (60−90 °C), 0:1 to 1:0] to obtain subfractions 2A−2N. Fractions 2D (11.2 mg), 2E (10.7 mg), and 2G (17.7 mg) were subjected to HPLC to afford compounds 3 (4.2 mg), 5 (2.6 mg), and 6 (1.5 mg), respectively. Subsequently, 3, 5, and 6 were subjected to chiral-phase HPLC using a CHIRALPAK AD-H column with a solvent of isopropanol−n-hexane (10:90) to afford 3a (tR 44.1 min), 3b (tR 49.7 min), 5a (tR 24.3 min), 5b (tR 42.2 min), 6a (tR 32.4 min), and 6b (tR 34.2 min), respectively. A Sephadex LH-20 column (MeOH) and a reversed-phase C18 silica gel column (H2O−MeOH, 7:3 to 0:1) were applied to separate fraction 2J (126.7 mg) and afforded subfractions J1 and J2. Subfraction J1 (48.0 mg) was purified by HPLC to obtain compounds 1 (3.9 mg) and 13 (3.1 mg). The enantiomers 1a (tR 16.7 min) and 1b (tR 19.2 min) were separated by chiral-phase HPLC using a CHIRALPAK AD-H column with a solvent of isopropanol−n-hexane (5:95). Calculation of the specific rotation of (+)-1a in MeOH was performed by the method of “self-consistent reaction field” (SCRF) at the B3LYP/6-31G (d)//CAM-B3LYP/631G+(d, p) level. Subfraction J2 (11.0 mg) was subjected to HPLC to obtain 2 (3.7 mg). The enantiomers 2a (tR 17.0 min) and 2b (tR 18.6 min) were isolated by chiral-phase HPLC using a CHIRALPAK AD-H column and eluting with isopropanol−n-hexane (15:85) as eluent. Fraction 2K (67.7 mg) was applied to a Sephadex LH-20 column (MeOH) as well as a reversed-phase C18 silica gel column (H2O− MeOH, 1:1 to 0:1) to yield subfractions K1 and K2. Compounds 7 (1.6 mg) and 8 (4.0 mg) were isolated by HPLC from subfraction K1 (35.0 mg). Subfraction K2 (21.0 mg) was subjected to HPLC to afford 4 (2.4 mg) and 18 (3.2 mg). Subsequently, compound 4 was separated by chiral-phase HPLC using a CHIRALPAK AD-H column with isopropanol−n-hexane (10:90) as eluent to afford 4a (tR 70.3 min) and 4b (tR 71.6 min). Moreover, the OR value of 4a was calculated by using the methodology described for 1. Compounds 11 (3.4 mg), 12 (8.2 mg), 14 (3.5 mg), and 20 (3.1 mg) were obtained from part of fraction 2L (88.8 mg) by using a Sephadex LH-20 column (MeOH), a reversed-phase C18 silica gel column (H2O−MeOH, 1:1 to 0:1), and HPLC in succession. Fraction 3 (1.4 g) was applied to a silica gel column [acetone−petroleum ether (60−90 °C), 0:1 to 1:0] to yield subfractions 3A−3K. Fraction 3C (199.6 mg) was purified by recrystallization from MeOH to yield 10 (100 mg). Fraction 3F (22.0 mg) and 3H (112.7 mg) were purified by HPLC to obtain 9 (0.9 mg) and 16 (44.5 mg), respectively. Fraction 3F (95.5 mg) was subjected to a Sephadex LH-20 column (CH2Cl2−MeOH = 1:1) to afford subfractions F1 and F2. Subsequently, subfractions F1 (36.0 mg) and F2 (11.0 mg) were subjected to HPLC to afford 17 (9.6 mg), 19 (3.8 mg), and 15 (2.1 mg). I



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NMR (CD4O, 100 MHz), see Table 3; HRESIMS m/z 297.1122 [M + H]+ (calcd 297.1121). Crystal Data for Compound 9. C18H16O4, M = 296.11, monoclinic, space group P21, α = 90.00°, β = 94.631(4)°, γ = 90.00°, a = 8.8218(12) Å, b = 11.2072(17) Å, c = 15.053(2) Å, V = 1483.4(4) Å3, Z = 44, μ(Cu Kα) = 0.711 mm−1, Dcalcd = 1.318 g/cm3, F(000) = 616. Totally 5086 reflections measured (2.715° ≤ 2θ ≤ 27.099°), 1766 parts were applied for calculations. The final R1 and wR2 were 0.259 (>2σ(I)) and 0.2114 (all data), respectively. The data of crystallography have been deposited with the Cambridge Crystallographic Data Centre with the number CCDC 1858356. The data can be found via www.ccdc.cam.ac.uk/products/csd/request. Cell Culture. The cancer cell lines NCI-H1299, A549, HepG-2, SMMC-7721, MCF-7, U251, Sw620, HT29, and KB were obtained from the China Academy of Sciences (China), Shanghai Institute for Biological Sciences (SIBS). The cell lines U251 and NCI-H1299 were cultured in DMEM medium, and other cell lines were cultured in RPMI 1640 medium, both of which were supplemented with penicillin G (100 U/mL), streptomycin (100 μg/mL), and 10% fetal bovine serum, and incubated at 37 °C in a humidified 5% CO2 incubator (Thermo Fisher Scientific). MTT Assay. A 96-well plate was used to seed the cells overnight. After incubation with several concentrations of the compounds to be tested for 48 h, MTT (5 mg/mL) was added to the cells and incubated for 4 h. The produced crystals were dissolved in DMSO. The cell viability was detected using a microplate reader at 570 nm (BioTek, USA). All experiments were performed three times. Flow Cytometry Assay. DCFH-DA (10 μM) was used in the measurement of ROS, Mito-Tracker Red (100 nM) for mitochondria integrity assays, JC-1 (5 μg/mL) for the assessment of MMP, and annexinV-FITC (0.1 mg/mL), and PI (0.5 mg/mL) for the assessment of apoptosis. The cells were examined by flow cytometry (Becton Dickinson, USA), and the data were analyzed using WinMDI 2.9 software. Western Blotting. A six-well plate was used to culture the cells overnight. The cells were exposed to various concentrations of 10 for 24 h. Western blotting was performed by using the methodology described previously.31,32 Live Cell Image. Cells seeded on coverslips were treated with 10 and incubated with Mito-Tracker Red (100 nM, Thermo Fisher, USA) at 37 °C for 30 min. After three washes with phosphate-buffered saline (PBS), the cells were incubated with 10 μg/mL Hoechst 33342 (Beyotime, China) for 15 min to mark nuclei. Fluorescence images were obtained using confocal microscopy (Carl Zeiss). Immunofluorescence Staining. After treatments, cells were fixed in 4% paraformaldehyde for 30 min and permeabilized by 0.3% Triton X-100 for 15 min. After three washes with PBS, the cells were blocked with 5% bovine serum albumin for 30 min. Cells were incubated with primary antibody HGMB1 (Abclonal, USA) at 4 °C overnight, then incubated with second antibody (ZSGB-BIO, China) at 37 °C for 1 h. Finally, cells were counter-stained with 2 mg/mL DAPI (Beyotime, China) for 15 min to mark nuclei. Fluorescence images were gained via using confocal microscopy (Carl Zeiss). Statistical Analysis. Data are presented as mean ± SD for triplicate experiments and analyzed by one-way ANOVA or Student’s t-test. A Pvalue < 0.05 was considered statistically significant.

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Article

AUTHOR INFORMATION

Corresponding Author

*Tel: +86-531-8838-2012. Fax: +86-531-8838-2019. E-mail: [email protected]. ORCID

Hong-Xiang Lou: 0000-0003-3300-1811 Author Contributions §

C.-Y. Zhang and Y. Gao contributed equally to this paper.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors gratefully acknowledge the National Natural Science Foundation of China (No. 81874293 and 81630093) for financial support.

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REFERENCES

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.8b00897. Calculation details, putative biosynthesis pathways of 1− 20, 1D, 2D NMR spectra, HRESIMS data, IR (KBr disc) and UV spectra of compounds 1−9, as well as the chiralphase column analysis, ECD spectrum of compound 1−7, as well as the supplementary data for biological activity (PDF) J



Article

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Prenylated Bibenzyls from the Chinese Liverwort Radula constricta

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